1. Field of the Invention
The invention relates to methods for the expression of human apolipoprotein(a) Kringle domains in bacterial cell hosts, to vectors useful for expression of the kringle domains and to the production of antibodies to human lipoprotein(a). Other aspects of the invention include methods of determining levels of serum lipoprotein(a) using these antibodies to define the antigenic distribution within the kringles 4 and 5 domains of human apo(a).
2. Description of Related Art
Human lipoprotein (a) has generated considerable interest because of its apparent correlation in blood with high risk of coronary heart disease (Scanu and Fless, 1990). Exactly how lipoliprotein (a) contributes to increased risk of heart disease is not known; however, according to some clinical studies, there appears to be a positive correlation between lipoprotein (a) blood levels and atherosclerosis. Lipoprotein (a) may favor the process of plaque buildup in the blood vessel wall. Indeed, lipoprotein (a) has been found at high levels in segments of coronary arteries after bypass surgery as well as in segments of peripheral vessels (Lawn, 1992).
Lipoprotein (a) is formed from the association of apo B100 and apolipoprotein (a) (apo (a) via a disulfide bond. Apo (a) is a large glycoprotein with extensive sequence homology to plasminogen (McLean, et al., 1987). Apo(a) exhibits size heterogeneity (300-800 KDa), the functional significance of which is not well understood. However, it is known that this size heterogeneity results from variation in the number of Kringle 4 domains in the molecule. Kringle 4 domains comprise on the average 78 amino acid residues with three highly conserved intramolecular disulfide bonds. It has been estimated that the number of Kringle 4-encoding repeats in the apo (a) gene can range from 9 to 35 (Lackner, et al., 1991). Kringle domains are so termed because of their resemblance to Danish pastries which have a comparable twisted structure (Lawn, 1992).
Kringle domains are found in other large proteins, most typically tissue plasminogen activator (Wilhelm, et al., 1990) and plasminogen (Mehnhart, et al., 1991). While there is extensive homology between apo (a) kringle 42 and kringle 4 of plasminogen, there are significant differences in the amino acid sequences, potentially causing changes in function. It has been suggested that physiologically lipoprotein (a) may effect the transport of cholesterol to damaged vessels, thus delivering a material critical for cell repair. On the other hand, excess lipoprotein (a) at the vessel wall site may favor accumulation of material, leading to atheroscelerotic plaque buildup (Lawn, 1992).
The Kringle 4 domains of apo (a) can be divided into 10 subtypes differing from plasminogen Kringle 4 by 12 to 23 amino acids (Morisett, et al., 1990). Like plasminogen, apo (a) shows high affinity for lysine-like ligands (Eaton, et al., 1987) and may also bind proline and hydroxyproline (Trieu, et al., 1991). However, the structural determinants of ligand binding for this protein are unknown. Individual Kringle domains appear to be independent structural units with autonomous functions (Trexler, et al., 1983). Kringle 2 of TPA and kringle 1 of plasminogen have been expressed in E. coli and found to bind to lysine, but kringle 4 and 5 domains from apo (a) have neither been expressed in E. coli nor characterized in specific binding properties.
In addition to kringle 4, apo (a) also contains a single Kringle 5 domain and a protease domain, both of which show high homology to the corresponding plasminogen domains (MacLean, et al., 1987).
Kringle domains of apo (a) may be the recognition sites for antibody binding. If such sites were identified, and were unique, a valuable method for specifically determining blood apo (a) levels would be available. At present, however, specific recognition sites on kringle 4 domains have not been identified and no truly immunospecific methods of determining apo (a) levels are available.
The present invention addresses one or more of the foregoing problems associated with the expression and purification of kringle apo (a) domains. The invention in particular includes the expression of recombinant kringle 4 and 5 domains from gram-negative bacteria, for example, E. coli. Recombinant kringle domains may be employed to generate antibodies which interact with apo (a) and lipoprotein (a). Such antibodies form the basis for various immunoassays designed to detect lipoprotein (a) and provide reproducible methods to detect this protein in human plasma.
One aspect of the present invention concerns the construction of an E. coli expression vector useful for producing recombinant kringle domains. These vectors include an inducible promoter sequence, a repressor gene sequence, a fusion protein and a polylinker gene sequence into which a DNA segment encoding a kringle polypeptide is cloned. The repressor gene is positioned upstream of an inducible promoter sequence which in turn is upstream of a selected fusion protein. The polylinker sequence is located between the fusion protein and the kringle polypeptide encoding DNA segments. The polylinker may be constructed with one or more restriction sites into which the kringle gene segment is inserted.
In preferred embodiments of the present invention, vectors incorporate maltose binding protein (MBP) as the fusion protein although other fusion partners might also be employed. MBP protein includes an amino acid sequence sensitive to Fxa protease. The expressed recombinant polypeptide includes three extraneous amino acids from the polylinker when the construct as shown in FIG. 1 is employed. MBP facilitates fusion protein isolation by virtue of its binding to amylose sepharose columns.
Kringle polypeptide expression has been demonstrated in E. coli but other prokaryotes or even eukaryotic host cells might be employed. Turning to the expression of the disclosed human apo (a) kringle polypeptides, once a suitable (full length if desired) clone or clones have been obtained, whether they be cDNA based or genomic, one may proceed to prepare an expression system for the recombinant preparation of the various regions or domains. The engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that either eukaryotic or prokaryotic expression systems may be employed in the expression of kringle apo (a) domains; however, vector constructs useful for such expression have not been heretofore available, nor has a recombinant apo (a) kringle been successfully expressed in a gram negative host cell until the present invention.
Human apo (a) kringle 4 and 5 domains have now been successfully expressed in bacterial expression systems with the production of correctly folded structures. The cDNA for apo (a) kringle and kringle 5 domains has been separately expressed in E. coli systems, with the encoded proteins being expressed as fusions with maltose binding protein (MBP), a most preferred embodiment. Other fusion proteins with such fusion partners as xcex2-galactosidase, ubiquitin, Schistosoma japonicum glutathione S-transferase, and the like are also envisioned. It is believed that bacterial expression has numerous advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained thereby.
If, however, an eukaryotic expression system is chosen, it is believed that almost any eukaryotic expression system may be utilized for the expression of human apo (a) kringles, e.g., baculovirus-based, glutamine synthase-based or dihydrofolate reductase-based systems could be employed. However, in preferred embodiments, it is contemplated that vectors constructed analogously to pIH821, will be employed incorporating an origin of replication and an efficient eukaryotic promoter, as exemplified by the eukaryotic vectors of the pCMV series, such as pCMV5. Examples of host cells commonly employed with expression include 239, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK, COS-7, RIN and MDCK cell lines.
For expression in this manner, one would position the coding sequences adjacent to and under the control of the promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the 5xe2x80x2 end of the transcription initiation site of the transcriptional reading frame of the protein between about 1 and about 50 nucleotides xe2x80x9cdownstreamxe2x80x9d of (i.e., 3xe2x80x2 of) the chosen promoter.
Where eukaryotic expression is contemplated, one will also typically desire to incorporate into the transcriptional unit which includes the kringle-encoding DNA, an appropriate polyadenylation site (e.g., 5xe2x80x2-AATAAA-3xe2x80x2) if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides xe2x80x9cdownstreamxe2x80x9d of the termination site of the protein at a position prior to transcription termination.
Whether employing a eukaryotic or a prokaryotic expression system, more than one kringle may be co-expressed in the same cell. This may be achieved by co-transfecting the cell with two distinct recombinant vectors, each bearing a copy of, for example, the kringle 4 or kringle 5-encoding DNA. Alternatively, a single recombinant vector may be constructed to include the coding regions for both domains, which could then be expressed in cells transfected with the single vector. In either event, the term xe2x80x9cco-expressionxe2x80x9d herein refers to the expression of more than one kringle domain in the same recombinant cell.
As used herein, the term xe2x80x9cengineeredxe2x80x9d or xe2x80x9crecombinantxe2x80x9d cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding a kringle domain has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a cDNA gene (i.e., they will not contain introns), a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.
Generally speaking, it is generally more convenient to employ as the recombinant gene a cDNA version of the gene. The use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect or transform the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. However, the inventor does not exclude the possibility of employing a genomic version of particular kringle genes where desired.
Where the introduction of a recombinant version of one or more of the foregoing genes is required, it will be important to introduce the gene such that it is under the control of a promoter that effectively directs the expression of the gene in the cell type chosen for engineering. In general, one will desire to employ a promoter that allows constitutive (constant) expression of the gene of interest. Commonly used constitutive promoters are generally viral in origin, and include the cytomegalovirus (CMV) promoter, the Rous sarcoma long-terminal repeat (LTR) sequence, and the SV40 early gene promoter. The use of these constitutive promoters will ensure a high, constant level of expression of the introduced genes. The inventor has noticed that the level of expression from the introduced gene(s) of interest can vary in different clones, probably as a function of the site of insertion of the recombinant gene in the chromosomal DNA. Thus, the level of expression of a particular recombinant gene can be chosen by evaluating different clones derived from each transfection or transformation experiment; once that line is chosen, the constitutive promoter ensures that the desired level of expression is permanently maintained. It may also be possible to use promoters that are specific for cell type used for engineering.
Monoclonal antibodies are prepared following in general the procedure of Goding (1980). This procedure is an immunization procedure using animals such as mice or rabbits. Purified kringle domains such as apo (a) 42 or kringle 5 domains are combined with DNA cellulose or similar material and taken up in Freunds complete adjuvant for the initial immunization. Subsequent immunizations typically utilize incomplete Freunds adjuvant. BALB/C mice, for example, may be employed, with an initial intraperitoneal immunization followed by intra-muscular injections. Blood samples are then periodically tested for the production of antibodies. High antibody titer animals are selected and the spleen is removed. Cells are isolated and tested for viability. Splenic lymphocytes are then fused with a non-secretor myeloma cell line such as P3-NS1-AG4-1 obtained from a commercial source using PEG to induce cells to fuse. Cells are plated out and media then used for inducing culture growth, typically such media as HAT medium. After two or more clonings, cells may be weaned from the growth medium onto serum.
Preliminary screening for antibodies may be accomplished by an ELISA method. Hybridoma screening kits may be used, (for example, BRL, Bethesda, Md.). Plates are coated with goat serum and hybridoma culture supernatant is added to control plates and to plates which have been coated with one or more of the kringle polypeptides. After suitable incubation, plates are washed. Beta-galactosidase conjugated to goat anti-mouse antibody is diluted 1 to 200 in PBS containing 1% goat serum added and further incubated. A chromofluoric substance is next added, for example, p-nitrophenyl phosphate and incubation continued, typically for about 1 hour. This is followed by quenching, for example, with a sodium carbonate solution, and wells are then read at 410 nm on an ELISA plate reader. Positive reaction is indicated by the development of a yellow color in the well.
Cells are cloned from positive wells by plating at 0.5 to 2 cells per well with later recloning at 0.3 to 0.5 cells per well. Positive clones are recognized by screening methods similar to those used for the hybridomas. Isotyping of cells may be conveniently performed using immunoglobin subtype identification. Kits supplying antigens for coating the plates are commercially available, such as affinity purified goat antimouse IgG heavy and light chain. Typical dilutions employed are about 1:50. The second antigen will be a kringle antigenic polypeptide. Once the hybridoma cells are successfully cloned they may be grown in bulk. Antibody concentration might be expected to range from 10 to 100 micrograms per milliliter of culture solution.
One may also employ the disclosed recombinant kringle polypeptides as antigenic substances as inoculums to generate antisera. Antibodies and antibody compositions may be isolated from the serum of immunized animals such as, rats, mice, rabbits, etc. Antibodies, whether monoclonals or polyclonals, generated from antisera or generated through hybridoma technology as noted above may then be used in the development of various diagnostic procedures.
Purified kringle domains obtained by the methods herein described may be employed to generate antibodies that are specific for lipoprotein (a). Lipoprotein (a) is found in human serum and is commonly thought to be associated with risk of coronary disease. Monoclonal antibodies directed specifically to lipoprotein (a) are expected to provide the selectivity and sensitivity necessary for developing immunobased assays to detect this protein, with numerous variations and modifications of well-known general types of immunoassays possible. Preferred assay methods of the invention include various types of enzyme-linked immunosorbant acids commonly known as ELISAS and well-known to the art; however, it will be appreciated that the utility of monoclonal antibodies directed to lipoprotein (a) is not limited to this particular type of assay and that other useful embodiments include RIAs and other non-enzyme linked antibody-binding assays or procedures.
In preferred ELISA type assays, lipoprotein (a) or convenient fragments of lipoprotein A are immobilized onto a selected surface, preferably a surface exhibiting protein affinity, such as the walls of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, a non-specific protein such as bovine serum albumin (BSA) or casein may be used to provide a complete coating on the selected surface material. This coating material should be antigenically neutral with regard to the test antisera. This provides blocking of non-specific adsorption sites on the immobilizing surface and reduces background caused by non-specific binding of the anti-lipoprotein (a).
Once binding of lipoprotein (a) to the well surface is accomplished and after coating with the non-reactive material and washing to remove unbound material, the immobilizing surface is contacted with an antibody specific for lipoprotein (a). The contacting is performed in such a manner as to be conducive to immuno complex formation. In this case the formation of a complex will be between lipoprotein (a) and a monoclonal antibody which is directed to an epitopic region of that protein. Conditions which facilitate formation of these sorts of complexes preferably include diluting the antibody with diluents such as BSA, bovine gamma-globulin and phosphate buffered saline/Tween. These added agents tend to assist in the reduction of non-specific background. After contact, the mixture is typically allowed to incubate for about two to four hours, preferably at a temperature from about 25 to 27xc2x0 C. Following incubation, the antibody antigen surface is washed in order to remove non-immuno complex material. A preferred washing procedure includes washing with solutions such as PBS/Tween or other appropriate buffers.
Following formation of specific immuno complexes between bound lipoprotein (a) and its monoclonal antibody and subsequent washing, the occurrence and amount of immuno complex formation may be determined by exposing the complex to a second antibody which has specificity for the first antibody. As the test sample will typically be of human origin, the second antibody will preferably be an antibody having specificity in general for human IgG. To provide detecting means, the second antibody will preferably have an associated enzyme that will generate color development upon incubation with an appropriate chromogenic substrate; thus, for example, one will desire to contact and incubate the antisera bound surface with a urease or peroxidase conjugated anti-human IgG for a period of time and under conditions which favor the development of immuno complex formation (e.g., incubation for two hours at room temperature in a PBS containing solution such as PBS/Tween).
After incubation with the second enzyme-tagged antibody and subsequent to washing for removing unbound material, the amount of enzyme label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or a 2,2xe2x80x2-azino dye (3-ethyl-benzthiazoline-6-sulfonic acid ABTS and H2O2). In the case of peroxidase as the enzyme label, quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectrophotometer.
As mentioned above, modification and changes may be made in the structure of apo (a) kringle polypeptides and still obtain a molecule having like or otherwise desirable characteristics. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein""s biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like or even countervailing properties (e.g., antagonistic v. agonistic). It is thus contemplated by the inventors that various changes may be made in the sequence of kringle 4 or kringle 5 apo (a) proteins or peptides (or underlying DNA) without appreciable loss of their biological utility or activity.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (xe2x88x920.4); threonine (xe2x88x920.7); serine (xe2x88x920.8); tryptophan (xe2x88x920.9); tyrosine (xe2x88x921.3); proline (xe2x88x921.6); histidine (xe2x88x923.2); glutamate (xe2x88x923.5); glutamine (xe2x88x923.5); aspartate (xe2x88x923.5); asparagine (xe2x88x923.5); lysine (xe2x88x923.9); and arginine (xe2x88x924.5).
It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules whether such molecules be enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid may be substituted by another amino acid having a similar hydropathic index and still obtain a biological functionally equivalent protein. In such changes, the substitution of amino acids whose hydropathic indices are within xc2x12 is preferred, those which are within xc2x11 are particularly preferred, and those within xc2x10.5 are even more particularly preferred.
Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0xc2x11); glutamate (+3.0xc2x11); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (xe2x88x920.5xc2x11); threonine (xe2x88x920.4); alanine (xe2x88x920.5); histidine (xe2x88x920.5); cysteine (xe2x88x921.0); methionine (xe2x88x921.3); valine (xe2x88x921.5); leucine (xe2x88x921.8); isoleucine (xe2x88x921.8); tyrosine (xe2x88x922.3); phenylalanine (xe2x88x922.5); tryptophan (xe2x88x923.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within xc2x12 is preferred, those which are within xc2x11 are particularly preferred, and those within xc2x10.5 are even more particularly preferred.
Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
Another aspect of the invention involves the preparation of antigenic portions of apo (a) kringle domains. The antigenic kringles, or epitopes of the desired antigen, are selected and a gene encoding that antigen or epitope region is inserted into one or more of the recombinant vectors disclosed. Appropriate host cells are transformed and after screening for transformants, one is selected that expresses the antigen or part of the antigen for which an antibody is desired.
In immunodiagnostics, it is often possible and indeed more practical to prepare antigens from segments of a known immunogenic protein or polypeptide. Certain epitopic regions may be used to produce responses similar to those produced by the entire antigenic polypeptide. Indeed, the inventor has shown that antibodies prepared from kringle 4 of human apo (a) react with human lipoprotein (a). Often, however, responses to epitopic regions are not so strong as responses to the entire polypeptide. It is sometimes useful in enhancing immunogenic response, particularly when trying to generate antibodies, to incorporate the kringle polypeptide or fragment incorporating an epitopic region with another protein. The inventor has found, for example, that kringle 4 fusion protein combined with maltose binding protein generates a good antibody response when injected into an animal, for example, a mouse. Thus similar responses will be expected for kringle 5. Because the kringle 5 is a unique segment, in contrast with the multiple repeats of kringle 4, antibodies generated to it or to its epitopes will likely be highly selective to human lipoprotein (a).